Sign up to receive free email alerts when patent applications with chosen keywords are publishedSIGN UP

Abstract:

Disclosed is a stable crystal of
1-(2'-cyano-2'-deoxy-β-D-arabinofuranosyl)cytosine
monohydrochloride. There is provided a crystal of
1-(2'-cyano-2'-deoxy-β-D-arabinofuranosyl)cytosine monohydrochloride
having characteristic peaks at 13.7°, 15.7°, 16.0°,
18.6°, 20.3°, and 22.7° as diffraction angles
(2θ±0.1°) measured by powder X-ray diffraction, and
having a melting point of 192° C. to 197° C.

Claims:

1. A crystal of 1-(2'-cyano-2'-deoxy-.beta.-D-arabinofuranosyl)cytosine
monohydrochloride having characteristic peaks at 13.7.degree.,
15.7.degree., 16.0.degree., 18.6.degree., 20.3.degree., and 22.7.degree.
as diffraction angles (20.+-.0.1.degree.) measured by powder X-ray
diffraction, and having a melting point of 192.degree. C. to 197.degree.
C.

2. A crystal of 1-(2'-cyano-2'-deoxy-.beta.-D-arabinofuranosyl)cytosine
monohydrochloride having characteristic peaks at 6.4.degree.,
12.6.degree., 17.3.degree., and 21.7.degree. as diffraction angles
(20.+-.0.1.degree.) measured by powder X-ray diffraction, and having a
melting point of 192.degree. C. to 197.degree. C.

3. A pharmaceutical composition comprising the crystal according to claim
1 or 2.

4. An anti-tumor agent comprising the crystal according to claim 1 or 2.

Description:

FIELD OF THE INVENTION

[0001] The subject invention relates to novel stable crystals of
1-(2'-cyano-2'-deoxy-β-D-arabinofuranosyl)cytosine
monohydrochloride, which are useful as a medicine showing an excellent
anti-tumor activity, and a pharmaceutical composition containing the
same.

BACKGROUND OF THE INVENTION

[0002] In general, when a compound is used as an effective active
ingredient of a pharmaceutical product, the compound is required to be
chemically and physically stabile so as to maintain a quality stably,
and/or to facilitate storage management. Therefore, the resulting
compound is preferably in the form of a stable crystal, and usually,
there are many instances in which ultrastable crystals are selected as
the drug substances for pharmaceutical products.

[0003] However, Patent Document 1, Non-Patent Document 1, and Non-Patent
Document 2 describe that
1-(2'-cyano-2'-deoxy-β-D-arabinofuranosyl)cytosine
monohydrochloride, which is a pyrimidine nucleoside derivative,
represented by the following formula (1):

##STR00001##

[0004] shows an in vitro suppressive effect for proliferation of human or
mouse tumor cells, and also shows an excellent anti-tumor activity in
vivo.

[0005] Methods for producing the compound have been reported, for example,
a method of dissolving
1-(2'-cyano-2'-deoxy-β-D-arabinofuranosyl)-N4-acetylcytosine
represented by the following formula (2):

##STR00002##

[0006] in a methanol solution of hydrochloric acid, allowing the compound
to react while stirring at room temperature, and after completion of the
reaction, crystallizing the product from ethanol and ether (Non-Patent
Documents 1 and 2); and a method of heating the compound represented by
the above formula (2) to reflux in acetic acid to thereby subject the
compound to de-N-acetylation, subsequently obtaining
1-(2'-cyano-2'-deoxy-β-D-arabinofuranosyl)cytosine represented by
the following formula (3):

##STR00003##

[0007] by silica gel column chromatography, dissolving the compound in a
methanol solution of hydrochloric acid, allowing the compound to react
while stirring at room temperature, and after completion of the reaction,
crystallizing the product from ethanol and ether (Patent Document 1).

[0008] The crystal obtainable by these methods was thought to be a 1/2
ethanolate at the time when the document was reported, and the melting
point of the crystal was considered to be 175° C. to 176°
C. (Patent Document 1 and Non-Patent Documents 1 and 2). However, other
than this melting point, no specific reports with regard to the crystal
polymorphism or stability have been reported.

[0012] The object of the subject invention is to provide stable crystals
of a compound that is useful as an anti-tumor agent.

Means for Solving the Problem

[0013] The inventors of the subject invention aimed to obtain a stable
crystal of 1-(2'-cyano-2'-deoxy-β-D-arabinofuranosyl)cytosine
monohydrochloride, and they first attempted to obtain a crystal of 1/2
ethanolate having a melting point of 175° C. to 176° C. by
the known methods described in Patent Document 1 and Non-Patent Documents
1 and 2.

[0014] However, unexpectedly, the inventors of the subject invention could
not reproduce the crystal of 1/2 ethanolate having a melting point of
175° C. to 176° C. described in the document (hereinafter,
also referred to as "crystal in known document") in an experiment
intended to reproduce the known methods described above.

[0015] Furthermore, the inventors of the subject invention strenuously
conducted the experiment in order to obtain the crystal of 1/2 ethanolate
having a melting point of 175° C. to 176° C., while taking
into consideration of various general conditions for crystal production
(see International Journal of Pharmaceutics, Vol. 60, 11 to 26 (1990),
and the like). However, the inventors eventually could not reproduce the
crystal described in known document.

[0016] In that case, it is contemplated that a crystal, that is not
obtainable even when such various production conditions are taken into
consideration, cannot be regarded as a preferable crystal that can be
produced and supplied stably in industrial scale. The reason may be that
the crystal obtained at that time may be a crystalline polymorph. When
the presence of a crystalline polymorph in a drug substance for
pharmaceutical products is confirmed, it is usually no longer easy to
stably produce a single crystal or a mixed type crystal in which the
content ratio is constantly maintained, and therefore, an extensive
investigation is required.

[0017] However, there have been some reports published in the past,
reporting that a crystal that could be conventionally obtained suddenly
cannot be obtained by the techniques of the related art ever since a
certain time. For example, in the case of Ritonavir, an anti-human
immunodeficiency virus (HIV) drug, a document reports that due to the
sudden appearance of Form II (a stable crystal), the traditionally
obtained Form I crystal can no longer be obtained by the traditional
production method (Organic Process Research and Development, Vol. 4, 413
to 417 (2000)). Also in the case of a compound that has been developed as
a cephem-based antibiotic substance, a document reports that due to the
sudden appearance of γ-crystal (a stable crystal), the
traditionally obtained α-crystal can no longer be obtained by the
traditional production method (Bunri Gijutsu (Separation Technology),
Vol. 33, 379 to 385 (2003)). Even in the case of a compound that has been
developed as a HIV-1 reverse transcriptase inhibitor, a document reports
that due to the sudden appearance of Form III crystal (a stable crystal),
the traditionally obtained Form I crystal can no longer be obtained by
the traditional production method (Organic Process Research and
Development, Vol. 9, 933 to 942 (2005)).

[0018] According to these findings, it is contemplated that the crystals
in prior art document obtainable at the relevant time could not be
obtained because the crystal in the prior art document was inferior to
the crystal of the subject invention in terms of stability. It is also
contemplated that, even if the crystal in prior art document were
temporarily generated in the reaction system, the crystal in the prior
art document immediately varies to a stable crystal through crystalline
transition, and consequently, the crystal in prior art document could not
be obtained.

[0019] During investigation for the subject invention, the inventors of
the subject invention found that, as shown in Examples described below,
crystals which do not contain ethanol and have a melting point 15°
C. to 20° C. higher than the melting point of 175° C. to
176° C. of the conventionally known crystal (hereinafter, also
referred to as "crystals of the subject invention") are obtained, and
that since the melting point of a crystal having excellent stability
(stable crystal) is generally higher than the melting point of an
unstable crystal (metastable crystal), the crystals of the subject
invention are stable crystals. Accordingly, the inventors have completed
the subject invention.

[0020] The subject invention relates to the following items (1) to (4).

[0021] (1) A crystal of
1-(2'-cyano-2'-deoxy-β-D-arabinofuranosyl)cytosine monohydrochloride
having characteristic peaks at 13.7°, 15.7°, 16.0°,
18.6°, 20.3°, and 22.7° as diffraction angles
(2θ±0.1°) measured by powder X-ray diffraction, and
having a melting point of 192° C. to 197° C.

[0022] (2) A crystal of
1-(2'-cyano-2'-deoxy-β-D-arabinofuranosyl)cytosine monohydrochloride
having characteristic peaks at 6.4°, 12.6°, 17.3°,
and 21.7° as diffraction angles (20±0.1°) measured by
powder X-ray diffraction, and having a melting point of 192° C. to
197° C.

[0023] (3) A pharmaceutical composition comprising the crystal according
to (1) or (2).

[0024] (4) An anti-tumor agent comprising the crystal according to (1) or
(2).

Effect of the Invention

[0025] Since the crystals of
1-(2'-cyano-2'-deoxy-β-D-arabinofuranosyl)cytosine monohydrochloride
of the subject invention show excellent physical stability and/or
chemical stability, the crystals are excellent as compared with other
amorphous form, other crystal forms or the like of
1-(2'-cyano-2'-deoxy-β-D-arabinofuranosyl)cytosine from the
viewpoints of, for example, storage stability, purity, usability (lower
hygroscopicity) and/or product manageability, and from the viewpoint that
the crystals have an excellent anti-tumor effect and are therefore useful
as anti-tumor agents. Thus, the crystals of the subject invention are
useful as a medicine.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG. 1 is a powder X-ray diffraction chart of the type I crystal.

[0029] The crystals of the subject invention, particularly three types of
crystals, such a type I crystal, a type II crystal and a type III
crystal, can be respectively obtained as single crystals by
crystallization or recrystallization from a solution containing
1-(2'-cyano-2'-deoxy-β-D-arabinofuranosyl)cytosine monohydrochloride
represented by the above formula (1) (hereinafter, also referred to as
"compound (1)").

[0030] Furthermore, the compound (1) may not be in a crystalline form,
also the compound (1) may be in a crystalline form. If the compound (1)
is in a crystalline form, for example, the type I crystal may be
separated by using the type II crystal as a raw material, or the type I
crystal may be separated by using the type III crystal as a raw material.

[0031] The 1-(2'-cyano-2'-deoxy-β-D-arabinofuranosylcytosine
monohydrochloride (compound (1)) used in the subject invention can be
obtained by an organic chemical synthesis method by using
1-(2'-cyano-2'-deoxy-β-D-arabinofuranosyl)-N4-acetylcytosine
represented by the above formula (2) (hereinafter, also referred to as
"compound (2)"), or
1-(2'-cyano-2'-deoxy-β-D-arabinofuranosyl)cytosine represented by
the above formula (3) (hereinafter, also referred to as "compound (3)")
as a raw material. For example, the compound (1) can be obtained by
subjecting the compound (2) to de-N-acetylation and addition of
hydrochloric acid, or by subjecting the compound (3) to addition of
hydrochloric acid.

[0032] More specific examples of the method for obtaining the compound (1)
include, for example, a method of treating the compound (2) with an acid
to obtain the compound (3), and subjecting this compound to addition of
hydrochloric acid to obtain the compound (1); a method of subjecting the
compound (2) to an acid treatment and addition of hydrochloric acid in
one step by using a hydrochloric acid-methanol solution, and thereby
obtaining the compound (1).

[0033] Among these, from the viewpoints of improving operating efficiency
or yield, it is preferable to carry out an acid treatment and
hydrochloric acid addition in one step, and the reaction condition of
this case is preferably using 1 to 20 mL of a 0.5% to 3% hydrochloric
acid-methanol solution relative to 100 mg of the compound (2), and
stirring the mixture at about 10° C. to 40° C. (preferably,
room temperature) for 0.5 to 3 hours (preferably 1±0.25 hours).

[0034] The compounds (2) and (3) can be produced by the production methods
described in the above mentioned Patent Document 1 and Non-Patent
Documents 1 and 2.

[0035] Furthermore, examples of the acid that is used in the acid
treatment include inorganic acids such as sulfuric acid and hydrochloric
acid; and organic acids such as acetic acid and trifluoroacetic acid.
These acids may be used alone or as mixtures of two or more kinds.

[0036] The method of deposition of the crystals of the subject invention
is not particularly limited, including, for example, a method of
dissolving the compound (1) in a solvent that dissolves the compound (1)
under heating (hereinafter, also referred to as "dissolving solvent"),
allowing the solution in which the compound (1) is dissolved to stand
still or cooling the solution with a cooling means, and thereby
depositing the crystal; a method of deposition of the crystal from the
solution in which the compound (1) is dissolved (hereinafter, also
referred to as "dissolved solution"), using a solvent having low
solubility for the compound (1) (hereinafter, also referred to as
"low-solubility solvent"). Among these, the method of deposition of the
crystal from the dissolving solution using a low-solubility solvent is
preferred. An example of this method may be, for example, a method of
adding the dissolved solution to a pre-cooled low-solubility solvent, and
thereby depositing the crystal.

[0037] The solvent used in the process of deposition of the crystal is not
particularly limited, such a solvent including, for example, water;
alcohols such as methanol, ethanol, and isopropanol; ketones such as
acetone and methyl ethyl ketone; ethers such as diethyl ether,
diisopropyl ether, and t-butyl methyl ether. These solvents can be used
alone or as mixtures of plural solvents.

[0038] The dissolving solvent is preferably a solvent that is capable of
dissolving the compound (1) under heating, and specifically, alcohols are
more preferred in view of stability. Among these, an alcohol having 1 to
5 carbon atoms, particularly ethanol, is preferred. Typically, the
temperature of the dissolving solvent when heating is preferably from
0° C. to the boiling point of the solvent, and more preferably
from 20° C. to 40° C. The concentration of the compound (1)
is not particularly limited, preferably 0.25 to 0.6 (W/V) % in the
dissolved solution.

[0039] The low-solubility solvent is preferably a solvent that is capable
of depositing the crystal when the solvent is added to the solution in
which the compound (1) is dissolved, and then the mixture is left to
stand or cooled. Specifically, ethers are preferred from the viewpoint of
stability, and among these, diethyl ether is particularly preferred.

[0040] When the dissolving solution is added, the temperature of the
low-solubility solvent is preferably from -40° C. to the boiling
point of the solvent, more preferably from -20° C. to 20°
C., and even more preferably from 0° C. to 10° C. On the
other hand, the temperature of the dissolving solution, when the
dissolving solution is added to the low-solubility solvent, is preferably
from -20° C. to the boiling point of the solvent, more preferably
from -5° C. to 45° C., and even more preferably from
0° C. to 40° C.

[0041] The amount of use of the low-solubility solvent is preferably from
0 to 1 part by volume relative to 1 part by volume of the dissolving
solution.

[0042] The temperature at the time of deposition is typically preferably
from -50° C. to 30° C., more preferably from -40° C.
to 20° C., and even more preferably from -30° C. to
10° C. In this case, the dissolving solution or a mixed solution
of the dissolving solution and the low-solubility solvent may be cooled
with the cooling means. For example, adding dropwise the dissolving
solution to a cooled vessel; cooling the dissolving solution or the mixed
solution may be carried out. In deposition, the solution may be left to
stand still, or may be stirred.

[0043] The deposited crystal can be isolated and purified from the
dissolving solution or the mixed solution by, for example, known
separation and purification techniques such as filtration, washing with
an organic solvent, and drying under reduced pressure. Examples of the
organic solvent used in the washing process include the low-solubility
solvents mentioned above, and among these, the ethers described above are
used with preference.

[0044] In this manner, the crystals of the subject invention, particularly
the type I crystal, type II crystal, and type III crystal, can be
produced.

[0045] For example, as a method for obtaining the type I crystal, it is
preferable to obtain the type I crystal by dissolving under heating the
compound (1) in ethanol, and then adding this solution dropwise into
diethyl ether that has been cooled to 0±5° C. while stirring
the mixture.

[0046] As a method for obtaining the type II crystal, it is preferable to
obtain the type II crystal by dissolving under heating the compound (1)
or the type I crystal in ethanol, subsequently cooling this solution to
40±5° C., and then adding diethyl ether dropwise to the
solution while stirring the mixture.

[0047] Furthermore, as a method for obtaining the type III crystal, it is
preferable to obtain the type III crystal by dissolving under heating the
compound (1) or the type I crystal in ethanol, and then adding this
solution dropwise onto a plate that has been cooled to 0±5° C.

[0048] The melting point of the type I to III crystals of the compound (1)
obtained as described above is 192° C. to 197° C., and as
shown in the powder X-ray diffraction charts of FIGS. 1 to 3, the
crystals are distinguished by the following characteristic diffraction
peaks.

[0049] That is, as shown in FIG. 1, the powder X-ray diffraction pattern
of the type I crystal exhibits characteristic peaks at around
13.7°, 15.7°, 16.0°, 18.6°, 20.3° and
22.7° as the diffraction angles (2θ±0.1°) measured
by powder X-ray diffraction.

[0052] The crystals of the subject invention show high storage stability,
are advantageous in terms of quality management, and also involve and
excellent usability. Particularly, as shown in the Examples described
below, even if the type I crystal is stored for a long time under high
temperature and high humidity conditions, the content of total analogues
is hardly detected, and the change of crystal form is hardly observed.
Also, since the type II crystal or the type III crystal is converted to
the type I crystal when the type II crystal or the type III crystal is
heated at around 70° C. to 90° C. for several hours and
stored at around 30° C. to 50° C. for a long time (one
month) and at a humidity of 75% RH or higher, the type I crystal has
superior stability as compared with the type II crystal or the type III
crystal.

[0053] As is obvious from the descriptions of the Patent Document 1, since
the compound (1) shows a strong anti-tumor activity, a pharmaceutical
composition containing the crystals of the subject invention can be used
particularly as an anti-tumor agent, and can be used for the manufacture
of the same.

[0054] The purity of the crystals of the subject invention, particularly
of the type I, type II and type III crystals in the respective crystal
forms of the same, is preferably substantially 95% or greater, more
preferably substantially 98% or greater, and even more preferably
substantially 99% or greater.

[0055] The crystals of the subject invention can be processed, after being
pulverized or without being pulverized, into various forms of anti-tumor
agents, for example, oral preparations such as tablets, capsules,
granules, fine granules, powders, and syrups; parenteral preparations
such as intravenous injectable preparations, subcutaneous injectable
preparations, intramuscular injectable preparations, and suppositories.
The crystals of the subject invention are to be intravenously
administered, and the dosage form is preferably an injectable
preparation. Such an injectable preparation is desirably used as a solid
injectable preparation, such as a lyophilized injectable preparation or a
powder injectable preparation, which can be used by dissolving at the
time of use.

[0056] The anti-tumor agent can be produced by a preparation method known
to and commonly used by those ordinarily skilled in the art, by using a
pharmaceutically acceptable carrier. In this case, other anti-tumor
agents, for example, 5-FU, a tegafur-uracil formulation, a
tegafur-gimeracil-oteracil potassium formulation, doxorubicin,
epirubicin, irinotecan hydrochloride, etoposide, docetaxel, pacritaxel,
cisplatin, carboplatin, oxaliplatin, krestin, lentinan, picibanil may
also be used in combination.

[0057] The dosage amount of the crystals of the subject invention in case
of using as an anti-tumor agent may vary depending on, for example, the
symptoms of the patient to whom the anti-tumor agent is applied, the
dosage form; however, in general, it is preferable to administer the
anti-tumor agent once or several times a day in an amount of 2.0 to 4.0
mg/m2 in terms of the crystals of the subject invention.

EXAMPLES

[0058] Hereinafter, the production method of the subject invention is
specifically described by way of Examples and Reference Examples.

Reference Example 1

Synthesis of
1-(2'-cyano-2'-deoxy-β-D-arabinofuranosyl)-N4-acetylcytosine

[0059] 1 M of THF solution (2 mL) of tetrabutylammonium fluoride and 0.06
mL (1 mmol) of acetic acid were added to a THF (5 mL) solution of
N4-acetyl-2'-cyano-2'-deoxy-3',5'-O-(1,1,3,3-tetraisopropyl
disiloxane-1,3-diyl)-1-β-D-arabinofuranosylcytosine (537 mg, 1
mmol), and the mixture was stirred at room temperature for 15 minutes.
After the stirring, the mixture was concentrated, and the residue was
purified by silica gel (11 g) column chromatography by using 8%
ethanol-chloroform as a developing solvent. The concentrated residue of
the target fraction was washed with hexane-diethyl ether, and thus 259 mg
(yield: 88%) of the title compound was obtained as a white crystal.

[0060] 1-(2'-cyano-2'-deoxy-β-D-arabinofuranosyl)-N4-acetylcytos-
ine (100 mg, 0.34 mmol) was dissolved in 1% hydrochloric acid-methanol
(7.5 mL), and the mixture was stirred at room temperature (20° C.
to 25° C.) for one hour. The reaction liquid was concentrated, and
ethanol (10 mL) was poured into this concentrate. The mixture was
co-evaporated, and thus 34 mg of the title compound was obtained (yield:
35%).

Preparation of Type I Crystal of
1-(2'-cyano-2'-deoxy-β-D-arabinofuranosyl)cytosine monohydrochloride

[0065] 1-(2'-cyano-2'-deoxy-β-D-arabinofuranosylcytosine
monohydrochloride (200 mg) obtained in Example 1 was heated to reflux in
ethanol (34 mL) and was dissolved therein. The solution was added
dropwise to diethyl ether (34 mL) that had been cooled to 0° C.
The mixture was stirred for 2 hours, and then a precipitate was collected
by filtration and was dried under reduced pressure at 35° C. Thus,
123 mg (yield: 62%) of the title compound was obtained as the type I
crystal.

[0072] The type I crystal of
1-(2'-cyano-2'-deoxy-β-D-arabinofuranosyl)cytosine monohydrochloride
(200 mg) was heated to reflux in ethanol (34 mL) and was dissolved
therein, and the solution was cooled to 40° C. Subsequently, the
ethanol (34 mL) in which this compound was dissolved was poured, by being
added dropwise, into diethyl ether (34 mL) which was in a reflux state
(reaction vessel heated to 40° C.). The mixture was stirred for
one hour while refluxing, and then a precipitate generated in diethyl
ether was collected by filtration and was dried under reduced pressure at
35° C. Thus, 119 mg (yield: 60%) of the title compound was
obtained as the II-type crystal.

[0073] FIG. 2 shows the powder X-ray diffraction chart of the type II
crystal obtained herein. This type II crystal was analyzed using a powder
X-ray diffraction apparatus in the same manner as described above, and
characteristic peaks were recognized at around 6.4°, 12.6°,
17.3°, and 21.7° as the diffraction angles
(20±0.1°) measured by powder X-ray diffraction.

[0078] The type I crystal of
1-(2'-cyano-2'-deoxy-β-D-arabinofuranosyl)cytosine monohydrochloride
(200 mg) was heated to reflux in ethanol (34 mL) and was dissolved
therein, and the solution thus obtained was slowly added dropwise to a
vessel that had been cooled to 0° C. The solution was stirred for
2 hours, and then a precipitate was collected by filtration and was dried
under reduced pressure at 35° C. Thus, 66 mg (yield: 33%) of the
title compound was obtained as the type III crystal.

[0079] FIG. 3 shows the powder X-ray diffraction chart of the type III
crystal obtained herein. This type III crystal was analyzed using a
powder X-ray diffraction apparatus in the same manner as described above,
and characteristic peaks were recognized at around 14.2°,
16.4°, 17.0°, 18.0°, and 20.2° as the
diffraction angles (20±0.1°) measured by powder X-ray
diffraction.

[0080] Melting point: 195° C.

[0081] Elemental Analysis:

[0082] Calculated value (as C10H13ClN4O4): C, 41.60;
H, 4.54; N,

[0083] Found value: C, 41.59; H, 4.42; N, 19.29

Example 5

Stability Test

[0084] The type I and type II crystal powders were respectively spread
uniformly on a glass petri dish, and an aluminum foil (or a transparent
resin film, for the purpose of storage under light exposure conditions)
which was appropriately perforated as vent holes therein was used as a
lid, to prepare test samples. These samples were respectively stored for
30 days under four kinds of conditions such as [60° C.],
[40° C./relative humidity (RH) 75%], [25° C./relative
humidity (RH) 60%, light exposure 2000 lxhr], and [25° C./relative
humidity (RH) 60%, light shading], and then the samples were tested using
high performance liquid chromatography (HPLC) and powder X-ray
diffraction (XRD).

[0085] For powder X-ray diffraction (XRD), the analysis was carried out as
described above.

[0086] For the HPLC analysis, 22.9 mg of the sample was dissolved in 10 mL
of 0.01 mol/L hydrochloric acid, and then the content of analogues in the
crystal was measured under the following conditions.

[0088] Mobile phase: 2.05 g of potassium dihydrogen phosphate is dissolved
in 3000 mL of water, and phosphoric acid is added to the solution to
adjust the pH to 3.0. The mobile phase is prepared by adding 60 mL of
methanol to 2940 mL of the solution liquid.

[0093] The term "total analogues" refers to the substances detected other
than 1-(2'-cyano-2'-deoxy-β-D-arabinofuranosyl)cytosine
monohydrochloride), and the term "total analogues (%)" refers to the
content ratio of total analogues with respect to the standard sample.

[0094] The results of this stability test are shown in Table 1.

[0095] The type I crystal and the type II crystal were both subjected to
the stability test under various conditions (under the conditions of
humidification and exposure). As a result, an increase in the total
analogues was not recognized as presented in Table 1, and the crystals
were found to be highly stable crystal forms.

[0096] Furthermore, the type II crystal was converted to the type I
crystal after 30 days under the conditions of 40° C./relative
humidity (RH) 75%, and the melting point and the powder X-ray diffraction
chart were consistent with those obtained in Example 1. Also, the type
III crystal was converted to the type I crystal when heated at 80°
C. for 2 hours, and the melting point and the powder X-ray diffraction
chart were consistent with those obtained in Example 1.

[0097] From these results, it was found that melting point of the type I
to III crystals are 192° C. to 197° C. and involve an
excellent stability. Particularly, the type I crystal was found to show
superior stability as compared with the type II crystal or the type III
crystal.

Patent applications in class Nitrogen, other than nitro or nitroso, bonded directly to the 4-position, and chalcogen bonded directly to the 2-position of the diazine ring (e.g., cytidines, etc.)

Patent applications in all subclasses Nitrogen, other than nitro or nitroso, bonded directly to the 4-position, and chalcogen bonded directly to the 2-position of the diazine ring (e.g., cytidines, etc.)